Summary G-quadruplexes (G4s) are non-canonical secondary structures in nucleic acids that are formed by guanine-rich sequences. G4 structural and functional studies have largely focused on DNA G4s, and the number of biological functions assigned to these motifs has grown rapidly since the discovery of their involvement in telomere biology. RNA G4s (RG4s) are less studied, but interest is increasing due to their association with multiple processes. A comprehensive understanding of how RNA G4s contribute to cell physiology and pathophysiology is the long-term research goal of the applicant. Multiple reports clearly demonstrate that G4s are enriched in mRNA 5?- and 3?-untranslated regulatory regions. There is an increasing evidence that RG4s control gene expression at transcriptional and post- transcriptional levels, although such data is largely based on in vitro studies. Testing the biological significance of RG4s requires proving that RG4s exist in vivo. Intriguingly, RG4s appear predominantly unfolded in eukaryotic cells, whereas they are readily folded in vitro, suggesting that in cells RG4s are constitutively recognized and actively unfolded. We hypothesize that RG4 folding-unfolding regulates mRNA homeostasis. This hypothesis is based on our analysis of human transcriptome that identifies RG4s as stress-responsive RNA elements. We will test this hypothesis with three specific aims. In AIM1, we will determine and characterize the fraction of the human transcriptome that contains putative stress-responsive RG4s in living cells. In AIM2, we will identify bona fide RG4-binding proteins using a novel approach based on proteomic/biochemical analysis of interactions between the 7-deazaguanine RNA derivatives and proposed binding factors. In AIM 3, we will use functional assays to determine the biological significance of RG4s in mRNA stability, localization and translation. We will use biophysical and biochemical methods to validate selected RG4 candidates. This work will elucidate how RG4-mediated functions contribute to cellular mRNA homeostasis, and will identify physiologically significant RG4-binding partners, which in turn may reveal molecular targets and pathways with therapeutic potential. The understanding of cellular functions of RG4 motifs is particularly relevant to the biology of cancer and neurodegeneration. RNA regions containing RG4s significantly overlap with regions containing disease- associated mutations. The proposed studies may elucidate molecular events underlying normal and pathological aspects of cell physiology, and identify events contributing to the tumorigenic or neurodegenerative changes.